Cloning and functional expression of the human GlcNAc-1-P transferase, the enzyme for the committed step of the dolichol cycle, by heterologous complementation in Saccharomyces cerevisiae.
The gene for the human dolichol cycle GlcNAc-1-P transferase (ALG7/GPT) was cloned by screening a human lung fibroblast cDNA library. The library was constructed in a Saccharomyces cerevisiae expression vector, and the positive clone was identified by complementation of the conditional lethal S.cerevisiae strain YPH-A7-GAL. This strain was constructed by replacing the endogenous promoter of the GPT-gene by the stringently regulated GAL1-promoter. This construct allows to specifically suppress the endogenous enzyme activity. The insert of the positive clone displayed an open reading frame of 1200 nucleotides, coding for a putative protein of 400 amino acids with a calculated molecular weight of 44.7 kDa. The deduced protein sequence shows a homology of over 90% when compared with other mammalian GPT sequences, thus resembling the close phylogenetic relationship between mammalian species. This homology however decreases to 40-50% when compared to more distantly related organisms such as S.cerevisiae , Schizosaccharomyces pombe , or Leishmania amazonensis . Biochemical characterization of the recombinant protein showed that it is functionally expressed in the S.cerevisiae strain YPH-A7-GAL. GlcNAc- and GlcNAc2-PP-Dolichol biosynthesis could be shown with isolated S.cerevisiae membranes from cells harboring the recombinant plasmid and grown on glucose thus suppressing transcription of the endogenous gene. Synthesis could be stimulated by dolicholphosphate and was inhibited by tunicamycin. These results show that we have cloned the human GlcNAc-1-P transferase by heterologous complementation in S. cerevisiae, a strategy that may be useful for the cloning and characterization of glycosyltransferases from a variety of organisms.
Eckert
V
V
Medizinisches Zentrum für Hygiene und Med. Mikrobiologie, Robert Koch Strasse 17, Philipps-Universität-Marburg, D-35037 Marburg, Germany.
Blank
M
M
Mazhari-Tabrizi
R
R
Mumberg
D
D
Funk
M
M
Schwarz
R T
RT
D000595:Amino Acid Sequence; D001483:Base Sequence; D003001:Cloning, Molecular; D017931:DNA Primers / Q000235:genetics; D018076:DNA, Complementary / Q000235:genetics; D004286:Dolichols / Q000096:biosynthesis; D004352:Drug Resistance, Microbial / Q000235:genetics; D015870:Gene Expression; D005816:Genetic Complementation Test; D006801:Humans; D008969:Molecular Sequence Data; D011995:Recombination, Genetic; D012441:Saccharomyces cerevisiae / Q000235:genetics; D017855:Transferases (Other Substituted Phosphate Groups) / Q000235:genetics*; D014415:Tunicamycin / Q000494:pharmacology
D017931:DNA Primers; D018076:DNA, Complementary; D004286:Dolichols; D014415:Tunicamycin; D017855:Transferases (Other Substituted Phosphate Groups); C023222:UDPacetylglucosamine-dolichyl-phosphate acetylglucosamine-1-phosphate transferase